1. Field of the Invention
The present invention relates generally to the fields of molecular biology, infectious diseases, and pharmacology. More particularly, it concerns methods of suppressing or preventing an infection of a subject or a cell by a pathogen using an MDA-7 polypeptide or a nucleic acid encoding an MDA-7 polypeptide.
2. Description of Related Art
Infections by pathogens are a major cause of morbidity and mortality in the U.S., and throughout the world. Successful vaccination programs against smallpox and polio, between 1950 and 1970, led to a general view by public health authorities, particularly in the West, that the war against infectious diseases was effectively over and some countries scaled-back health measures. However, the emergence of HIV and multi-resistant organisms has shown that constant vigilance is needed where pathogens are concerned.
In addition, in light of the events of Sep. 11, 2001, many anti-terrorist experts and government officials believe that the prospect of a bioterrorism attack against the United States using pathogens is highly likely. For example, terrorists may have the ability of gaining access to pathogens that are associated with substantial morbidity and mortality. Large scale release of a highly infectious pathogenic agent could infect the inhabitants of targeted cities and other areas. The attacks could result in long- and short-term health consequences and possibly death. Thus, in addition to the continuing need for the possibility of mass treatment of pathogens, there is an urgent need to develop strategies to prevent the morbidity and mortality associated with a bioterrorist attack wherein the public is exposed to deadly pathogens.
Viruses pose the threat of inflicting serious morbidity and mortality on the population. As obligate intracellular parasites, viruses rely exclusively on the translational machinery of the host cell for the synthesis of viral proteins. This relationship has imposed numerous challenges on both the infecting virus and the host cell. Importantly, viruses must compete with the endogenous transcripts of the host cell for the translation of viral mRNA. Eukaryotic viruses have evolved diverse mechanisms to ensure translational efficiency of viral mRNA above and beyond that of cellular RNA. These mechanisms serve to redirect the translational apparatus to favor viral transcripts, and they often come at the expense of the host cell.
One such mechanism whereby viruses ensure translational efficiency involves the cellular kinase known as interferon-induced, double-stranded (ds) RNA-activated serine/threonine protein kinase (PKR). PKR is involved in the regulated of apoptosis, cell-proliferation, signal transduction, and differentiation of cells. PKR is believed to localize in the cytoplasm and nucleolus of the cell, and associates with ribosomes. Overexpression or activation of PKR in HeLa, COS-1, U937, and NIH-3T3 cells has been shown to lead to apoptosis. In addition, mouse embryo fibroblasts from PKR knock-out mice are resistant to apoptotic cell death in response to dsRNA, tumor necrosis factor, and lipopolysaccharide. RNaseL and PKR mediate the IFN-induced antiviral response of the host, which is required to limit viral protein synthesis and pathogenesis. As part of the innate immune response to viral infection, RNaseL and PKR are activated by dsRNAs produced as intermediates in viral replication.
PKR also mediates the antiviral actions of interferon, in part by phosphorylating the alpha subunit of eukaryotic initiation factor 2 (eIF-2α), resulting in acute inhibition of mRNA translation and a concomitant block in viral replication (Merrick and Hershey, 1996; Meurs et al., 1990). In addition, PKR facilitates IFN-induced transcriptional programs by participating in the activation of nuclear factor kappa B and INF-regulatory factor 1 (Kumar et al., 1997).
Many viruses inhibit PKR activity. One such example is Hepatitis C virus (HCV), a member of the Flaviviridae, which mediates persistent infection within a majority of infected individuals. Many strains of HCV are resistant to alpha interferon (IFN) therapy. It has recently been shown that HCV represses PKR function through the actions of the viral NS5A protein, which binds and inhibits PKR in vivo (Gale et al., 1998, Gale et al., 1999). Other examples of viruses that inhibit PKR activity include adenovirus, EBV, poxvirus, influenza virus, reovirus, HIV, polio, HSV, and SV40 (Gale et al., 2000). Inhibition of PKR results in inhibition of apoptosis, with resulting continued translation of viral proteins.
Another cellular regulatory mechanism involved in protecting against viral infection involves the PRK-like endoplasmic-reticulum (ER)-resident kinase (PERK). PERK is a type I transmembrane protein that attenuates protein synthesis during endoplasmic reticulum (ER) stress by phosphorylating serine 51 on eIF2α. The HCV envelope protein, E2, can serve as a pseudosubstrate inhibitor of PERK and a potential viral regulator of the ER stress response (Pavio et al., 2003).
Another mediator of ER stress is the Unfolded Protein Response (UPR) (Gething and Sambrook, 1992). The UPR is an ER-to-nucleus signal transduction pathway that regulates a wide variety of target genes and is responsible for maintaining cellular homeostasis. Transient stresses such as glucose deprivation or perturbation of calcium or redox homeostasis can result in transient inhibition of protein translation and growth arrest. Prolonged UPR activation leads to activation of death-related signaling pathways and ultimately, to apoptotic death (Kaufman, 2002; Ron, 2002). The UPR is used by many endoplasmic reticulum-tropic viruses, such as HCV, flaviviruses, HHV6, rubella, LCMV, HIV, and Hepatitis B virus (HBV), to facilitate their life cycle and pathogenesis. Note that many viruses, especially positive strand viruses, modify host intracellular membranes to create a suitable compartment for viral replication—this requirement for virus-induced intracellular membrane production results in activation of UPR.
It is clear that pathogens other than viruses can activate the PKR and UPR defense systems. For example, PKR is activated in response to Salmonella infection (Shtrichman et al., 2002). Shtrichman et al. (2002) describes tissue-selectivity of interferon-stimulated gene expression in mice infected with Dam (+) versus Dam (−) Salmonella enterica serovar Typhimurium strains. In addition, the UPR serves as a defense system in plants (Oh et al., 2003). Oh et al. (2003) have shown conservation between animals and plants of the cis-activating element involved in the UPR.
The intracellular mediators, IRE1 and PERK are activated by ER stress, which can be induced by high-level glycoprotein expression. All enveloped viruses produce excess glycoproteins that could elicit PERK and IRE1 activation to meet the need for increased folding and secretory capacity. Thus, UPR activation is a common feature of viral replication.
Therefore, there is the need for novel agents that can be applied in the prevention and treatment of infections of subjects by viruses and other pathogens. Such novel methods may target not only immunostimulation, but may also be directed at interfering with viral replication through activation of PKR and the UPR response. These agents can be applied in ameliorating the effects of bioterrorism attacks wherein the public is exposed to pathogens.